RESUMEN
Glycerophospholipids have emerged as a significant contributor to the intracellular growth of pathogenic protist Toxoplasma gondii. Phosphatidylserine (PtdSer) is one such lipid, attributed to the locomotion and motility-dependent invasion and egress events in its acutely infectious tachyzoite stage. However, the de novo synthesis of PtdSer and the importance of the pathway in tachyzoites remain poorly understood. We show that a base-exchange-type PtdSer synthase (PSS) located in the parasite's endoplasmic reticulum produces PtdSer, which is rapidly converted to phosphatidylethanolamine (PtdEtn) by PtdSer decarboxylase (PSD) activity. The PSS-PSD pathway enables the synthesis of several lipid species, including PtdSer (16:0/18:1) and PtdEtn (18:2/20:4, 18:1/18:2 and 18:2/22:5). The PSS-depleted strain exhibited a lower abundance of the major ester-linked PtdEtn species and concurrent accrual of host-derived ether-PtdEtn species. Most phosphatidylthreonine (PtdThr) species-an exclusive natural analog of PtdSer, also made in the endoplasmic reticulum-were repressed. PtdSer species, however, remained largely unaltered, likely due to the serine-exchange reaction of PtdThr synthase in favor of PtdSer upon PSS depletion. Not least, the loss of PSS abrogated the lytic cycle of tachyzoites, impairing the cell division, motility, and egress. In a nutshell, our data demonstrate a critical role of PSS in the biogenesis of PtdSer and PtdEtn species and its physiologically essential repurposing for the asexual reproduction of a clinically relevant intracellular pathogen.
Asunto(s)
Retículo Endoplásmico , Toxoplasma , Toxoplasma/enzimología , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/enzimología , Humanos , Fosfatidilserinas/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , CarboxiliasasRESUMEN
Phospholipids, the most abundant membrane lipid components, are crucial in maintaining membrane structures and homeostasis for biofunctions. As a structurally diverse and tightly regulated system involved in multiple organelles, phospholipid metabolism is complicated to manipulate. Thus, repurposing phospholipids for lipid-derived chemical production remains unexplored. Herein, we develop a Saccharomyces cerevisiae platform for de novo production of oleoylethanolamide, a phospholipid derivative with promising pharmacological applications in ameliorating lipid dysfunction and neurobehavioral symptoms. Through deregulation of phospholipid metabolism, screening of biosynthetic enzymes, engineering of subcellular trafficking and process optimization, we could produce oleoylethanolamide at a titer of 8,115.7 µg l-1 and a yield on glucose of 405.8 µg g-1. Our work provides a proof-of-concept study for systemically repurposing phospholipid metabolism for conversion towards value-added biological chemicals, and this multi-faceted framework may shed light on tailoring phospholipid metabolism in other microbial hosts.
Asunto(s)
Endocannabinoides/biosíntesis , Ingeniería Metabólica/métodos , Ácidos Oléicos/biosíntesis , Fosfolípidos/metabolismo , Saccharomyces cerevisiae/metabolismo , Acilcoenzima A/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Coenzima A Ligasas/genética , Endocannabinoides/genética , Enzimas/genética , Enzimas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Fúngica de la Expresión Génica , Glucosa/metabolismo , Lisofosfolipasa/genética , Lisofosfolipasa/metabolismo , Microorganismos Modificados Genéticamente , Monoacilglicerol Lipasas/genética , Monoacilglicerol Lipasas/metabolismo , Ácidos Oléicos/genética , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/metabolismo , Fosfolípidos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Phosphatidylserine (PS), synthesized in the endoplasmic reticulum (ER) by phosphatidylserine synthase (PSS), is transported to the plasma membrane (PM) and mitochondria through distinct routes. The in vivo functions of PS at different subcellular locations and the coordination between different PS transport routes are not fully understood. Here, we report that Drosophila PSS regulates cell growth, lipid storage and mitochondrial function. In pss RNAi, reduced PS depletes plasma membrane Akt, contributing to cell growth defects; the metabolic shift from phospholipid synthesis to neutral lipid synthesis results in ectopic lipid accumulation; and the reduction of mitochondrial PS impairs mitochondrial protein import and mitochondrial integrity. Importantly, reducing PS transport from the ER to PM by loss of PI4KIIIα partially rescues the mitochondrial defects of pss RNAi. Together, our results uncover a balance between different PS transport routes and reveal that PSS regulates cellular homeostasis through distinct metabolic mechanisms.
Asunto(s)
CDP-Diacilglicerol-Inositol 3-Fosfatidiltransferasa/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Retículo Endoplásmico/metabolismo , Fosfatidilserinas/metabolismo , Animales , Homeostasis , Mitocondrias/metabolismo , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Salinity-induced lipid alterations have been reported in many plant species; however, how lipid biosynthesis and metabolism are regulated and how lipids work in plant salt tolerance are much less studied. Here, a constitutively much higher phosphatidylserine (PS) content in the plasma membrane (PM) was found in the euhalophyte Salicornia europaea than in Arabidopsis. A gene encoding PS synthase (PSS) was subsequently isolated from S. europaea, named SePSS, which was induced by salinity. Multiple alignments and phylogenetic analysis suggested that SePSS belongs to a base exchange-type PSS, which localises to the endoplasmic reticulum. Knockdown of SePSS in S. europaea suspension cells resulted in reduced PS content, decreased cell survival rate, and increased PM depolarization and K+ efflux under 400 or 800 mM NaCl. By contrast, the upregulation of SePSS leads to increased PS and phosphatidylethanolamine levels and enhanced salt tolerance in Arabidopsis, along with a lower accumulation of reactive oxygen species, less membrane injury, less PM depolarization and higher K+/Na+ in the transgenic lines than in wild-type (WT). These results suggest a positive correlation between PS levels and plant salt tolerance, and that SePSS participates in plant salt tolerance by regulating PS levels, hence PM potential and permeability, which help maintain ion homeostasis. Our work provides a potential strategy for improving plant growth under multiple stresses.
Asunto(s)
CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/fisiología , Membrana Celular/fisiología , Chenopodiaceae/enzimología , Proteínas de Plantas/fisiología , Arabidopsis , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Membrana Celular/metabolismo , Chenopodiaceae/genética , Chenopodiaceae/metabolismo , Chenopodiaceae/fisiología , Retículo Endoplásmico/enzimología , Técnicas de Silenciamiento del Gen , Fosfatidilserinas/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Estrés Salino , Tolerancia a la Sal , Alineación de SecuenciaRESUMEN
Phospholipids are an integral part of the cellular membrane structure and can be produced by a de novo biosynthetic pathway and, alternatively, by the Kennedy pathway. Studies in several yeast species have shown that the phospholipid phosphatidylserine (PS) is synthesized from CDP-diacylglycerol and serine, a route that is different from its synthesis in mammalian cells, involving a base-exchange reaction from preexisting phospholipids. Fungal-specific PS synthesis has been shown to play an important role in fungal virulence and has been proposed as an attractive drug target. However, PS synthase, which catalyzes this reaction, has not been studied in the human fungal pathogen Cryptococcus neoformans Here, we identified and characterized the PS synthase homolog (Cn Cho1) in this fungus. Heterologous expression of Cn CHO1 in a Saccharomyces cerevisiae cho1Δ mutant rescued the mutant's growth defect in the absence of ethanolamine supplementation. Moreover, an Sc cho1Δ mutant expressing Cn CHO1 had PS synthase activity, confirming that the Cn CHO1 encodes PS synthase. We also found that PS synthase in C. neoformans is localized to the endoplasmic reticulum and that it is essential for mitochondrial function and cell viability. Of note, its deficiency could not be complemented by ethanolamine or choline supplementation for the synthesis of phosphatidylethanolamine (PE) or phosphatidylcholine (PC) via the Kennedy pathway. These findings improve our understanding of phospholipid synthesis in a pathogenic fungus and indicate that PS synthase may be a useful target for antifungal drugs.
Asunto(s)
Cryptococcus neoformans/metabolismo , Retículo Endoplásmico/metabolismo , Viabilidad Microbiana , Fosfatidilserinas/biosíntesis , Animales , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Cryptococcus neoformans/genética , Citidina Difosfato Diglicéridos/genética , Citidina Difosfato Diglicéridos/metabolismo , Retículo Endoplásmico/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Fosfatidilserinas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Phosphatidylserine synthase (Pss) is involved in the metabolic pathway in phospholipid synthesis in different organisms. In this study, Pss expression in Vibrio parahaemolyticus was verified through liquid chromatography tandem-mass spectrometry. To analyze the characteristics of Pss, the recombinant Pss was overexpressed and purified from Escherichia coli. The optimum temperature and pH of Pss were 40 °C and 8, respectively. When reacting with divalent metal, Pss activity decreased. In addition, Pss could not only use cytidine diphosphate diacylglycerol (CDP-DAG, 16:0), but also CDP-DAG (18:1) as a substrate to produce cytidine 5'-monophosphate. Furthermore, the 3D structure of Pss was predicted, and the results revealed that histidine and lysine of the two HKD motifs were present in the catalytic site. Moreover, CDP-DAG (16:0) was docked with the Pss model. To investigate whether the two HKD motifs in Pss are important to its activity, site-directed mutagenesis of histidine was performed. The results revealed that the activities of both H131A and H352A were diminished. Little is known regarding the catalytic site of type I Pss. This is the first report on the biochemical characterization of Pss in V. parahaemolyticus.
Asunto(s)
Proteínas Bacterianas/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Vibrio parahaemolyticus/enzimología , Proteínas Bacterianas/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , Cromatografía Liquida , Escherichia coli/genética , Histidina/genética , Concentración de Iones de Hidrógeno , Mutagénesis Sitio-Dirigida , Fosfolípidos/metabolismo , Espectrometría de Masas en Tándem , Temperatura , Vibrio parahaemolyticus/genéticaRESUMEN
The outer leaflet of the viral membrane of Ebola virus (EBOV) virions is enriched with phosphatidylserine (PtdSer), which is thought to play a central role in viral tropism, entry, and virus-associated immune evasion. We investigated the effects of inhibiting synthesis and/or export of PtdSer to the cell surface of infected cells on viral infectivity. Knockdown of both PtdSer synthase enzymes, PTDSS1 and PTDSS2, effectively decreased viral production. Decreased PtdSer expression resulted in an accumulation of virions at the plasma membrane and adjacent of intracellular organelles, suggesting that virion budding is impaired. The addition of inhibitors that block normal cellular trafficking of PtdSer to the plasma membrane resulted in a similar accumulation of virions and reduced viral replication. These findings demonstrate that plasma membrane-associated PtdSer is required for efficient EBOV budding, increasing EBOV infectivity, and could constitute a potential therapeutic target for the development of future countermeasures against EBOV.
Asunto(s)
Ebolavirus/patogenicidad , Fosfatidilserinas/fisiología , Animales , Transporte Biológico , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Chlorocebus aethiops , Células Vero , Virión/fisiología , Liberación del Virus , Replicación ViralRESUMEN
The PAH1-encoded phosphatidate phosphatase (PAP), which catalyzes the committed step for the synthesis of triacylglycerol in Saccharomyces cerevisiae, exerts a negative regulatory effect on the level of phosphatidate used for the de novo synthesis of membrane phospholipids. This raises the question whether PAP thereby affects the expression and activity of enzymes involved in phospholipid synthesis. Here, we examined the PAP-mediated regulation of CHO1-encoded phosphatidylserine synthase (PSS), which catalyzes the committed step for the synthesis of major phospholipids via the CDP-diacylglycerol pathway. The lack of PAP in the pah1Δ mutant highly elevated PSS activity, exhibiting a growth-dependent up-regulation from the exponential to the stationary phase of growth. Immunoblot analysis showed that the elevation of PSS activity results from an increase in the level of the enzyme encoded by CHO1 Truncation analysis and site-directed mutagenesis of the CHO1 promoter indicated that Cho1 expression in the pah1Δ mutant is induced through the inositol-sensitive upstream activation sequence (UASINO), a cis-acting element for the phosphatidate-controlled Henry (Ino2-Ino4/Opi1) regulatory circuit. The abrogation of Cho1 induction and PSS activity by a CHO1 UASINO mutation suppressed pah1Δ effects on lipid synthesis, nuclear/endoplasmic reticulum membrane morphology, and lipid droplet formation, but not on growth at elevated temperature. Loss of the DGK1-encoded diacylglycerol kinase, which converts diacylglycerol to phosphatidate, partially suppressed the pah1Δ-mediated induction of Cho1 and PSS activity. Collectively, these data showed that PAP activity controls the expression of PSS for membrane phospholipid synthesis.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Regulación Fúngica de la Expresión Génica , Modelos Biológicos , Fosfatidato Fosfatasa/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/química , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , Eliminación de Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosfatidato Fosfatasa/química , Fosfatidato Fosfatasa/genética , Fosfolípidos/metabolismo , Regiones Promotoras Genéticas , Transporte de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Elementos de Respuesta , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genéticaRESUMEN
Protein kinase C in Saccharomyces cerevisiae, i.e., Pkc1, is an enzyme that plays an important role in signal transduction and the regulation of lipid metabolic enzymes. Pkc1 is structurally similar to its counterparts in higher eukaryotes, but its requirement of phosphatidylserine (PS) and diacylglycerol (DAG) for catalytic activity has been unclear. In this work, we examined the role of these lipids in Pkc1 activity with protein and peptide substrates. In agreement with previous findings, yeast Pkc1 did not require PS and DAG for its activity on the peptide substrates derived from lipid metabolic proteins such as Pah1 [phosphatidate (PA) phosphatase], Nem1 (PA phosphatase phosphatase), and Spo7 (protein phosphatase regulatory subunit). However, the lipids were required for Pkc1 activity on the protein substrates Pah1, Nem1, and Spo7. Compared with DAG, PS had a greater effect on Pkc1 activity, and its dose-dependent interaction with the protein kinase was shown by the liposome binding assay. The Pkc1-mediated degradation of Pah1 was attenuated in the cho1Δ mutant, which is deficient in PS synthase, supporting the notion that the phospholipid regulates Pkc1 activity in vivo.
Asunto(s)
Diglicéridos/metabolismo , Metabolismo de los Lípidos/genética , Fosfatidato Fosfatasa/metabolismo , Proteína Quinasa C/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Péptidos/metabolismo , Fosfatidato Fosfatasa/genética , Fosfatidilserinas/metabolismo , Fosforilación , Proteína Quinasa C/genética , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , Especificidad por Sustrato , Triglicéridos/metabolismoRESUMEN
Close contacts between organelles, often called membrane contact sites (MCSs), are regions where lipids are exchanged between organelles. Here, we identify a novel mechanism by which cells promote phospholipid exchange at MCSs. Previous studies have shown that phosphatidylserine (PS) synthase activity is highly enriched in portions of the endoplasmic reticulum (ER) in contact with mitochondria. The objective of this study was to determine whether this enrichment promotes PS transport out of the ER. We found that PS transport to mitochondria was more efficient when PS synthase was fused to a protein in the ER at ER-mitochondria contacts than when it was fused to a protein in all portions of the ER. Inefficient PS transport to mitochondria was corrected by increasing tethering between these organelles. PS transport to endosomes was similarly enhanced by PS production in regions of the ER in contact with endosomes. Together, these findings indicate that PS production at MCSs promotes PS transport out of the ER and suggest that phospholipid production at MCSs may be a general mechanism of channeling lipids to specific cellular compartments.
Asunto(s)
CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , Retículo Endoplásmico/metabolismo , Metabolismo de los Lípidos/genética , Fosfatidilserinas/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Bacterianas/genética , Transporte Biológico/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Membrana Celular/química , Membrana Celular/enzimología , Retículo Endoplásmico/enzimología , Endosomas/metabolismo , Escherichia coli/enzimología , Glicosiltransferasas/genética , Lipogénesis/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Membranas Mitocondriales/enzimología , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Phosphatidylserine (PS) synthase (Cho1p) and the PS decarboxylase enzymes (Psd1p and Psd2p), which synthesize PS and phosphatidylethanolamine (PE), respectively, are crucial for Candida albicans virulence. Mutations that disrupt these enzymes compromise virulence. These enzymes are part of the cytidine diphosphate-diacylglycerol pathway (i.e. de novo pathway) for phospholipid synthesis. Understanding how losses of PS and/or PE synthesis pathways affect the phospholipidome of Candida is important for fully understanding how these enzymes impact virulence. The cho1Δ/Δ and psd1Δ/Δ psd2Δ/Δ mutations cause similar changes in levels of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol and PS. However, only slight changes were seen in PE and phosphatidylcholine (PC). This finding suggests that the alternative mechanism for making PE and PC, the Kennedy pathway, can compensate for loss of the de novo synthesis pathway. Candida albicans Cho1p, the lipid biosynthetic enzyme with the most potential as a drug target, has been biochemically characterized, and analysis of its substrate specificity and kinetics reveal that these are similar to those previously published for Saccharomyces cerevisiae Cho1p.
Asunto(s)
CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Candida albicans/enzimología , Candida albicans/metabolismo , Fosfolípidos/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , Candida albicans/genética , Eliminación de Gen , Cinética , Especificidad por SustratoRESUMEN
The phospholipid (PL) requirement in fish is revealed by enhanced performance when larvae are provided PL-enriched diets. To elucidate the molecular mechanism underlying PL requirement in Atlantic salmon, Salmo salar, were fed a minimal PL diet and tissue samples from major lipid metabolic sites were dissected from fry and parr. In silico analysis and cloning techniques demonstrated that salmon possess a full set of enzymes for the endogenous production of PL. The gene expression data indicated that major PL biosynthetic genes of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn) and phosphatidylinositol (PtdIns) display lower expression in intestine during the early developmental stage (fry). This is consistent with the hypothesis that the intestine of salmon is immature at the early developmental stage with limited capacity for endogenous PL biosynthesis. The results also indicate that intact PtdCho, PtdEtn and PtdIns are required in the diet at this stage. PtdCho and sphingomyelin constitute the predominant PL in chylomicrons, involved in the transport of dietary lipids from the intestine to the rest of the body. As sphingomyelin can be produced from PtdCho in intestine of fry, our findings suggest that supplementation of dietary PtdCho alone during early developmental stages of Atlantic salmon would be sufficient to promote chylomicron formation. This would support efficient transport of dietary lipids, including PL precursors, from the intestine to the liver where biosynthesis of PtdEtn, PtdSer, and PtdIns is not compromised as in intestine facilitating efficient utilisation of dietary energy and the endogenous production of membrane PL for the rapidly growing and developing animal.
Asunto(s)
CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Grasas de la Dieta/metabolismo , Proteínas de Peces/metabolismo , Salmo salar/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , Quilomicrones/biosíntesis , Grasas de la Dieta/administración & dosificación , Proteínas de Peces/genética , Regulación del Desarrollo de la Expresión Génica , Mucosa Intestinal/metabolismo , Intestinos/crecimiento & desarrollo , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Fosfatidilcolinas/biosíntesis , Fosfatidiletanolaminas/biosíntesis , Fosfatidilinositoles/biosíntesis , Salmo salar/genética , Salmo salar/crecimiento & desarrollo , Alineación de Secuencia , Esfingomielinas/biosíntesisRESUMEN
Microbial secretion is integral for regulating cell homeostasis as well as releasing virulence factors during infection. The genes encoding phosphatidylserine synthase (CHO1) and phosphatidylserine decarboxylase (PSD1 and PSD2) are Candida albicans genes involved in phospholipid biosynthesis, and mutations in these genes affect mitochondrial function, cell wall thickness, and virulence in mice. We tested the roles of these genes in several agar-based secretion assays and observed that the cho1Δ/Δ and psd1Δ/Δ psd2Δ/Δ strains manifested less protease and phospholipase activity. Since extracellular vesicles (EVs) are surrounded by a lipid membrane, we investigated the effects of these mutations on EV structure, composition, and biological activity. The cho1Δ/Δ mutant releases EVs comparable in size to wild-type EVs, but EVs from the psd1Δ/Δ psd2Δ/Δ strain are much larger than those from the wild type, including a population of >100-nm EVs not observed in the EVs from the wild type. Proteomic analysis revealed that EVs from both mutants had a significantly different protein cargo than that of EVs from the wild type. EVs were tested for their ability to activate NF-κB in bone marrow-derived macrophage cells. While wild-type and psd1Δ/Δ psd2Δ/Δ mutant-derived EVs activated NF-κB, the cho1Δ/Δ mutant-derived EV did not. These studies indicate that the presence and absence of these C. albicans genes have qualitative and quantitative effects on EV size, composition, and immunostimulatory phenotypes that highlight a complex interplay between lipid metabolism and vesicle production.
Asunto(s)
Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/metabolismo , Candida albicans/genética , Candida albicans/metabolismo , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Lípidos/genética , Animales , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Carboxiliasas/genética , Carboxiliasas/metabolismo , Línea Celular , Pared Celular/genética , Pared Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Macrófagos/microbiología , Ratones , Proteómica/métodos , Virulencia/genéticaRESUMEN
Lipid metabolism in Trypanosoma brucei, the causative agent of African sleeping sickness, differs from its human host in several fundamental ways. This has lead to the validation of a plethora of novel drug targets, giving hope of novel chemical intervention against this neglected disease. Cytidine diphosphate diacylglycerol (CDP-DAG) is a central lipid intermediate for several pathways in both prokaryotes and eukaryotes, being produced by CDP-DAG synthase (CDS). However, nothing is known about the single T. bruceiâ CDS gene (Tb927.7.220/EC 2.7.7.41) or its activity. In this study we show TbCDS is functional by complementation of a non-viable yeast CDS null strain and that it is essential in the bloodstream form of the parasite via a conditional knockout. The TbCDS conditional knockout showed morphological changes including a cell-cycle arrest due in part to kinetoplast segregation defects. Biochemical phenotyping of TbCDS conditional knockout showed drastically altered lipid metabolism where reducing levels of phosphatidylinositol detrimentally impacted on glycoylphosphatidylinositol biosynthesis. These studies also suggest that phosphatidylglycerol synthesized via the phosphatidylglycerol-phosphate synthase is not synthesized from CDP-DAG, as was previously thought. TbCDS was shown to localized the ER and Golgi, probably to provide CDP-DAG for the phosphatidylinositol synthases.
Asunto(s)
CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Citidina Difosfato/metabolismo , Diglicéridos/metabolismo , Trypanosoma brucei brucei/enzimología , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , Ciclo Celular , Retículo Endoplásmico/enzimología , Eliminación de Gen , Prueba de Complementación Genética , Aparato de Golgi/enzimología , Metabolismo de los Lípidos , Fosfatidilinositoles/análisis , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
Phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of cell membranes to generate and maintain phospholipid asymmetry. The genome of budding yeast encodes four heteromeric flippases (Drs2p, Dnf1p, Dnf2p, and Dnf3p), which associate with the Cdc50 family noncatalytic subunit, and one monomeric flippase Neo1p. Flippases have been implicated in the formation of transport vesicles, but the underlying mechanisms are largely unknown. We show here that overexpression of the phosphatidylserine synthase gene CHO1 suppresses defects in the endocytic recycling pathway in flippase mutants. This suppression seems to be mediated by increased cellular phosphatidylserine. Two models can be envisioned for the suppression mechanism: (i) phosphatidylserine in the cytoplasmic leaflet recruits proteins for vesicle formation with its negative charge, and (ii) phosphatidylserine flipping to the cytoplasmic leaflet induces membrane curvature that supports vesicle formation. In a mutant depleted for flippases, a phosphatidylserine probe GFP-Lact-C2 was still localized to endosomal membranes, suggesting that the mere presence of phosphatidylserine in the cytoplasmic leaflet is not enough for vesicle formation. The CHO1 overexpression did not suppress the growth defect in a mutant depleted or mutated for all flippases, suggesting that the suppression was dependent on flippase-mediated phospholipid flipping. Endocytic recycling was not blocked in a mutant lacking phosphatidylserine or depleted in phosphatidylethanolamine, suggesting that a specific phospholipid is not required for vesicle formation. These results suggest that flippase-dependent vesicle formation is mediated by phospholipid flipping, not by flipped phospholipids.
Asunto(s)
Fosfatidilserinas/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Endosomas/metabolismo , Proteínas de Transferencia de Fosfolípidos/genética , Transporte de Proteínas , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are metabolically related membrane aminophospholipids. In mammalian cells, PS is required for targeting and function of several intracellular signaling proteins. Moreover, PS is asymmetrically distributed in the plasma membrane. Although PS is highly enriched in the cytoplasmic leaflet of plasma membranes, PS exposure on the cell surface initiates blood clotting and removal of apoptotic cells. PS is synthesized in mammalian cells by two distinct PS synthases that exchange serine for choline or ethanolamine in phosphatidylcholine (PC) or PE, respectively. Targeted disruption of each PS synthase individually in mice demonstrated that neither enzyme is required for viability whereas elimination of both synthases was embryonic lethal. Thus, mammalian cells require a threshold amount of PS. PE is synthesized in mammalian cells by four different pathways, the quantitatively most important of which are the CDP-ethanolamine pathway that produces PE in the ER, and PS decarboxylation that occurs in mitochondria. PS is made in ER membranes and is imported into mitochondria for decarboxylation to PE via a domain of the ER [mitochondria-associated membranes (MAM)] that transiently associates with mitochondria. Elimination of PS decarboxylase in mice caused mitochondrial defects and embryonic lethality. Global elimination of the CDP-ethanolamine pathway was also incompatible with mouse survival. Thus, PE made by each of these pathways has independent and necessary functions. In mammals PE is a substrate for methylation to PC in the liver, a substrate for anandamide synthesis, and supplies ethanolamine for glycosylphosphatidylinositol anchors of cell-surface signaling proteins. Thus, PS and PE participate in many previously unanticipated facets of mammalian cell biology. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.
Asunto(s)
Membrana Celular/metabolismo , Citidina Difosfato/análogos & derivados , Etanolaminas/metabolismo , Mitocondrias/metabolismo , Fosfatidiletanolaminas/biosíntesis , Fosfatidilserinas/biosíntesis , Animales , Ácidos Araquidónicos/metabolismo , Transporte Biológico , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Carboxiliasas/metabolismo , Citidina Difosfato/metabolismo , Descarboxilación , Endocannabinoides/metabolismo , Retículo Endoplásmico/metabolismo , Metilación , Ratones , Ratones Noqueados , Fosfatidilcolinas/metabolismo , Alcamidas Poliinsaturadas/metabolismoRESUMEN
Systemic infections by Candida spp. are associated with high mortality rates, partly due to limitations in current antifungals, highlighting the need for novel drugs and drug targets. The fungal phosphatidylserine synthase, Cho1, from Candida albicans is a logical antifungal drug target due to its importance in virulence, absence in the host, and conservation among fungal pathogens. Inhibitors of Cho1 could serve as lead compounds for drug development, so we developed a target-based screen for inhibitors of purified Cho1. This enzyme condenses serine and cytidyldiphosphate-diacylglycerol (CDP-DAG) into phosphatidylserine (PS) and releases cytidylmonophosphate (CMP). Accordingly, we developed an in vitro nucleotidase-coupled malachite-green-based high throughput assay for purified C. albicans Cho1 that monitors CMP production as a proxy for PS synthesis. Over 7,300 molecules curated from repurposing chemical libraries were interrogated in primary and dose-responsivity assays using this platform. The screen had a promising average Z' score of ~0.8, and seven compounds were identified that inhibit Cho1. Three of these, ebselen, LOC14, and CBR-5884, exhibited antifungal effects against C. albicans cells, with fungicidal inhibition by ebselen and fungistatic inhibition by LOC14 and CBR-5884. Only CBR-5884 showed evidence of disrupting in vivo Cho1 function by inducing phenotypes consistent with the cho1∆∆ mutant, including a reduction of cellular PS levels. Kinetics curves and computational docking indicate that CBR-5884 competes with serine for binding to Cho1 with a Ki of 1,550 ± 245.6 nM. Thus, this compound has the potential for development into an antifungal compound. IMPORTANCE: Fungal phosphatidylserine synthase (Cho1) is a logical antifungal target due to its crucial role in the virulence and viability of various fungal pathogens, and since it is absent in humans, drugs targeted at Cho1 are less likely to cause toxicity in patients. Using fungal Cho1 as a model, there have been two unsuccessful attempts to discover inhibitors for Cho1 homologs in whole-cell screens prior to this study. The compounds identified in these attempts do not act directly on the protein, resulting in the absence of known Cho1 inhibitors. The significance of our research is that we developed a high-throughput target-based assay and identified the first Cho1 inhibitor, CBR-5884, which acts both on the purified protein and its function in the cell. This molecule acts as a competitive inhibitor with a Ki value of 1,550 ± 245.6 nM and, thus, has the potential for development into a new class of antifungals targeting PS synthase.
Asunto(s)
Antifúngicos , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa , Candida albicans , Inhibidores Enzimáticos , Candida albicans/efectos de los fármacos , Candida albicans/enzimología , Candida albicans/genética , Antifúngicos/farmacología , Antifúngicos/química , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Ensayos Analíticos de Alto Rendimiento , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/química , Pruebas de Sensibilidad Microbiana , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/química , Fosfatidilserinas/metabolismo , Furanos , TiofenosRESUMEN
Numb regulates endocytosis in many metazoans, but the mechanism by which it functions is not completely understood. Here we report that the Caenorhabditis elegans Numb ortholog, NUM-1A, a regulator of endocytic recycling, binds the C isoform of transbilayer amphipath transporter-1 (TAT-1), a P4 family adenosine triphosphatase and putative aminophospholipid translocase that is required for proper endocytic trafficking. We demonstrate that TAT-1 is differentially spliced during development and that TAT-1C-specific splicing occurs in the intestine where NUM-1A is known to function. NUM-1A and TAT-1C colocalize in vivo. We have mapped the binding site to an NXXF motif in TAT-1C. This motif is not required for TAT-1C function but is required for NUM-1A's ability to inhibit recycling. We demonstrate that num-1A and tat-1 defects are both suppressed by the loss of the activity of PSSY-1, a phosphatidylserine (PS) synthase. PS is mislocalized in intestinal cells with defects in tat-1 or num-1A function. We propose that NUM-1A inhibits recycling by inhibiting TAT-1C's ability to translocate PS across the membranes of recycling endosomes.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Adenosina Trifosfatasas/metabolismo , Animales , Sitios de Unión , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Endocitosis/genética , Endocitosis/fisiología , Endosomas/genética , Endosomas/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transferencia de Fosfolípidos/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas , Transporte de ProteínasRESUMEN
Sphingolipids play critical roles in many physiologically important events in the yeast Saccharomyces cerevisiae. In this study, we found that csg2Δ mutant cells defective in the synthesis of mannosylinositol phosphorylceramide exhibited abnormal intracellular accumulation of an exocytic v-SNARE, Snc1, under phosphatidylserine synthase gene (PSS1)-repressive conditions, although in wild-type cells, Snc1 was known to cycle between plasma membranes and the late Golgi via post-Golgi endosomes. The mislocalized Snc1 was co-localized with an endocytic marker dye, FM4-64, upon labelling for a short time. The abnormal distribution of Snc1 was suppressed by deletion of GYP2 encoding a GTPase-activating protein that negatively regulates endosomal vesicular trafficking, or expression of GTP-restricted form of Ypt32 GTPase. Furthermore, an endocytosis-deficient mutant of Snc1 was localized to plasma membranes in PSS1-repressed csg2Δ mutant cells as well as wild-type cells. Thus, the PSS1-repressed csg2Δ mutant cells were indicated to be defective in the trafficking of Snc1 from post-Golgi endosomes to the late Golgi. In contrast, the vesicular trafficking pathways via pre-vacuolar endosomes in the PSS1-repressed csg2Δ mutant cells seemed to be normal. These results suggested that specific complex sphingolipids and phosphatidylserine are co-ordinately involved in specific vesicular trafficking pathway.
Asunto(s)
Endosomas/metabolismo , Fosfatidilserinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Esfingolípidos/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Membrana Celular/metabolismo , Endocitosis/fisiología , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Fosfatidilserinas/química , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esfingolípidos/químicaRESUMEN
In vascular plants, the regulation of stem cell niche determines development of aerial shoot which consists of stems and lateral organs. Intercalary meristem (IM) controls internode elongation in rice and other grasses, however little attention has been paid to the underlying mechanism of stem cell maintenance. Here, we investigated the stem development in rice and showed that the Shortened Uppermost Internode 1 (SUI1) family of genes are pivotal for development of rice stems. We demonstrated that SUI-family genes regulate the development of IM for internode elongation and also the cell expansion of the panicle stem rachis in rice. The SUI-family genes encoded base-exchange types of phosphatidylserine synthases (PSSs), which possessed enzymatic activity in a yeast complementary assay. Overexpression of SUI1 and SUI2 caused outgrowths of internodes during vegetative development, and we showed that expression patterns of Oryza Sativa Homeobox 15 (OSH15) and Histone4 were impaired. Furthermore, genome-wide gene expression analysis revealed that overexpression and RNA knockdown of SUI-family genes affected downstream gene expression related to phospholipid metabolic pathways. Moreover, using Ultra-performance liquid chromatography-quadrupole time of flight-mass spectrometry, we analyzed PS contents in different genetic backgrounds of rice and showed that the quantity of very long chain fatty acids PS is affected by transgene of SUI-family genes. Our study reveals a new mechanism conveyed by the SUI1 pathway and provides evidence to link lipid metabolism with plant stem cell maintenance.